AB5075, a Highly Virulent Isolate of Acinetobacter baumannii, as a Model Strain for the Evaluation of Pathogenesis and Antimicrobial Treatments

Abstract

Acinetobacter baumannii is recognized as an emerging bacterial pathogen because of traits such as prolonged survival in a desiccated state, effective nosocomial transmission, and an inherent ability to acquire antibiotic resistance genes. A pressing need in the field of A. baumannii research is a suitable model strain that is representative of current clinical isolates, is highly virulent in established animal models, and can be genetically manipulated. To identify a suitable strain, a genetically diverse set of recent U.S. military clinical isolates was assessed. Pulsed-field gel electrophoresis and multiplex PCR determined the genetic diversity of 33 A. baumannii isolates. Subsequently, five representative isolates were tested in murine pulmonary and Galleria mellonella models of infection. Infections with one strain, AB5075, were considerably more severe in both animal models than those with other isolates, as there was a significant decrease in survival rates. AB5075 also caused osteomyelitis in a rat open fracture model, while another isolate did not. Additionally, a Tn5 transposon library was successfully generated in AB5075, and the insertion of exogenous genes into the AB5075 chromosome via Tn7 was completed, suggesting that this isolate may be genetically amenable for research purposes. Finally, proof-of-concept experiments with the antibiotic rifampin showed that this strain can be used in animal models to assess therapies under numerous parameters, including survival rates and lung bacterial burden. We propose that AB5075 can serve as a model strain for A. baumannii pathogenesis due to its relatively recent isolation, multidrug resistance, reproducible virulence in animal models, and genetic tractability.

Importance: The incidence of A. baumannii infections has increased over the last decade, and unfortunately, so has antibiotic resistance in this bacterial species. A. baumannii is now responsible for more than 10% of all hospital-acquired infections in the United States and has a >50% mortality rate in patients with sepsis and pneumonia. Most research on the pathogenicity of A. baumannii focused on isolates that are not truly representative of current multidrug-resistant strains isolated from patients. After screening of a panel of isolates in different in vitro and in vivo assays, the strain AB5075 was selected as more suitable for research because of its antibiotic resistance profile and increased virulence in animal models. Moreover, AB5075 is susceptible to tetracycline and hygromycin, which makes it amenable to genetic manipulation. Taken together, these traits make AB5075 a good candidate for use in studying virulence and pathogenicity of this species and testing novel antimicrobials.

FIG 1

Pulsed-field gel electrophoresis of A. baumannii strains. Genomic DNA was isolated from 33 A. baumannii clinical isolates, digested with ApaI, and separated by pulsed-field gel electrophoresis (PFGE). Patterns of electrophoresis were compared using BioNumerics 6.0 software. The ICC was determined by multiplex PCR analysis, and brackets delineate the approximate grouping of each strain.

FIG 2

Survival of Galleria mellonella larvae infected with A. baumannii. Kaplan-Meier survival curves of G. mellonella infected with 1.0 × 10⁵ CFU of selected strains of A. baumannii are shown. Curves were compared via the Mantel-Cox test with the Bonferroni correction for multiple comparisons. AB5075 showed significantly increased mortality compared to AB4857, AB5711, and AB0057 (P < 0.0125).

FIG 3

Assessment of A. baumannii virulence with the mouse pulmonary model. Kaplan-Meier survival curves of mice infected with 5.0 × 10⁶ CFU of selected A. baumannii strains. Curves were compared via the Mantel-Cox test with the Bonferroni correction for multiple comparisons. AB5075 showed significantly increased mortality compared to AB4857, AB5711, AB0057, and AB5256 (P < 0.0125).

FIG 4

Bacterial levels in lung tissue in the mouse pulmonary model. Box-and-whisker plots of CFU burdens in lungs are shown for days 2 and 3 postinfection. Boxes show median and interquartile ranges, while whiskers represent the 95% confidence interval CI. Strains were compared via the Kruskal-Wallis test followed by Dunn’s multiple comparisons posttests. * and ***, P < 0.05 and 0.001, respectively.

FIG 5

Rifampin as proof of concept in the G. mellonella model. Kaplan-Meier survival curves of G. mellonella infected with 6.0 × 10⁵ CFU of AB5075 are shown. Worms received a single treatment, 30 min postinfection, of DMSO, 5 mg/kg rifampin, or 10 mg/kg rifampin. Curves were compared via the Mantel-Cox test. The control-treated worms showed significantly increased mortality compared to rifampin-treated worms (P < 0.001).

FIG 6

Rifampin as proof of concept in the mouse lung model. (A) Kaplan-Meier survival curves of mice infected with 5.0 × 10⁶ CFU of selected strains of A. baumannii are shown. Mice were treated once daily IP with 0, 2.5, 5, or 10 mg/kg rifampin. Curves were compared via the Mantel-Cox test. Rifampin treatments of 5 and 10 mg/kg resulted in significantly increased survival compared to the untreated control (P = 0.0035 and 0.0008, respectively). (B) Box-and-whisker plots of CFU burdens within lungs are shown for day 2 postinfection. Boxes show median and interquartile ranges, while whiskers represent 95% CI. Treatments were compared via the Kruskal-Wallis test followed by Dunn’s multiple comparisons posttests. * and ***, P < 0.05 and 0.001, respectively.